IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 1.1

Experiment

Aims

• To determine if construct expresses in vitro at temperatures of: 4oC, 15oC, 25oC, 30oC, 37oC, 50oC
• To determine how long the system can last (when GFP is no longer produced) at each temperature.
• To determine the maximum rate of GFP produced at each temperature range by taking fluoresence reading over a period of time ( time frame to be determined by Friday 17th August)

Status
???
Equipments

• Fluorometer + PC
• Fridge at 4oC
• Water bath in cold room at 15oC
• 30oC water bath
• 37oC shaking incubator
• 50oC heating block
• 6 Fluorometer plates (black)
• 6 Sealing plate mats
• Gilson pipettes 1000, 200 and 20
• Eppendorf Tubes
• Plate Centrifuge
• Stopwatch

Reagents

• Our Prepared S30 extract
• Commercial S30 E.coli extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• Commercial S30 T7 extract. Including:
  • 175µl Amino Acid Mixture Minus Cysteine, 1mM
  • 175µl Amino Acid Mixture Minus Methionine, 1mM
  • 175µl Amino Acid Mixture Minus Leucine, 1mM
  • 450µl T7 S30 Extract, Circular (3 × 150µl)
  • 750µl S30 Premix Without Amino Acids
• MiiA water x1ml
• GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)

Steps